Project Summary Hsp104 is a hexameric AAA+ protein disaggregase from yeast that can rapidly disassemble disordered aggregates, preamyloid oligomers, amyloids, and prions. These activities have allowed yeast to harness beneficial prions for adaptive purposes. However, humans and metazoan lack a direct Hsp104 homologue, and are vulnerable to protein misfolding, which underpins several fatal neurodegenerative diseases including Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). We previously engineered potentiated Hsp104 variants that antagonize misfolding of several proteins associated with neurodegenerative disease (TDP-43 implicated in ALS and ?Syn implicated in PD). However, these variants lack substrate specificity and can be toxic in some circumstances. Thus, understanding how Hsp104 selects substrates remains an important objective. Hsp104 homologues are found in all nonmetazoan eukaryotes and eubacteria. However, the vast majority of these homologues remain unexplored. I have established that Hsp104 homologues from diverse lineages, including protozoa, fungi, and plants, are selective modifiers of TDP-43 and ?Syn proteotoxic misfolding. Based on my preliminary findings, I hypothesize that differences in substrate recognition and binding among Hsp104 homologues underpins their substrate selectivity. To test this hypothesis, I will leverage molecular evolution algorithms to identify sequence motifs that modulate Hsp104 substrate-selectivity. I will use pure protein biochemistry to evaluate the ability of substrate-selective Hsp104 orthologues and engineered variants to disaggregate protein aggregates in vitro, and to gain direct mechanistic insight into and define parameters of Hsp104 substrate selection. Finally, I will evaluate the therapeutic potential of the selective Hsp014s I identify and engineer by (1) introducing the ?Syn-selective Hsp104s into a C. elegans model of Parkinson's disease and monitoring protection of dopaminergic neurons in the worm and (2) introducing TDP-43-selective Hsp104s into a D. melanogaster model of TDP-43-opathy and monitoring the lifespan and motor neuron function of lies. These studies constitute an important step toward evolving enhanced disaggregases to combat protein misfolding in neurodegenerative disease.